1. Field of the Invention
The present invention relates to a human lens epithelial cell line and a production method thereof.
2. Description of Related Art
Various studies have been made to clarify cataract which is the main cause of blindness. A truly efficacious agent for prophylaxis and treatment of cataract has not been developed, and the most effective therapy is currently a surgical extraction of an opacified lens and implantation of an artificial lens (intraocular lens). Artificial lenses are prepared from polymethylmethacrylate, silicone and the like, and may lack bioavailability and biocompatibility, causing ocular inflammation, edema and the like at graft site. Such artificial lenses are under development, which includes, for example, monofocal lenses and multifocal lenses capable of acquiring sight on both far view and near view. Nonetheless, artificial lenses are markedly different in precision, shape and the like from inborn lenses having authentic amplitude of accommodation.
While artificial lenses prepared from inborn materials may be of interest, the peculiarity of the lens in that it is composed of transparent and refractive tissues has hindered the development of artificial lenses made from inborn materials.
Under the circumstances, the present inventor took note of grafting human-originated lens epithelial cells in place of an artificial lens and allowing proliferation and differentiation of said cells at the graft site, thereby to regenerate human lens to cure cataract.
The lens consists of a single cell species inclusive of lens epithelial cells and lens fiber cells resulting from differentiation and elongation of the lens epithelial cells, with its entirety covered by a lens capsule made by collagen in the main. Inasmuch as the lens fiber cells have been known to have lost proliferation potency and disintegrates DNA and nucleus as they become fibrous, lens epithelial cells alone can be actually grafted for regeneration of a lens. The lens epithelial cells of many animal species have been known to differentiate into lens fiber cells in vitro, and have been used for elucidation of differentiation mechanism of cells and studies of cataract.
The human lens epithelial cells have noticeably low proliferation potency when compared to lens epithelial cells of animals such as cow and chicken. Furthermore, lens epithelial cells obtained from adult cataract patients or senile cataract patients are either substantially incapable of culture or barely proliferated even if they permit culture at all. While the lens epithelial cells obtained from infants can be cultured rather easily and the cultured cells can be subcultured several times, their proliferation potency is limited and cytomegalic cells and cell degeneration in a long-term subculture have been reported Reddy V. N. et al., Exp. Eye Res., vol. 47, pp. 465-478 (1988), Ibaraki N. et al., Invest. Ophthalmol. Vis. Sci., vol. 36, pp. 2304-2312 (1995)!. Consequently, they are not suitable for practical use.
In view of this, it has been tried to make a human lens epithelial cell line. However, spontaneous human lens epithelial cell line has not been obtained. In the meantime, a human lens epithelial cell line has been successfully prepared by infecting human lens epithelial cells with adenovirus 12 in which large T antigen gene derived from simian virus 40 (SV40) known as an immortalizing gene has been incorporated Andley U. P. et al., Invest. Ophthalmol. Vis. Sci., vol. 35, pp. 3094-3102 (1994)!. This method leads to the production of viruses from said cell line, since cells are infected with a live virus for the introduction of the immortalizing gene. It is an undeniable fact that a patient may be infected with the virus when such cells are grafted, even if the lens should be successfully regenerated.
In this method, moreover, a high efficiency of virus infection makes it difficult to isolate a cell line from a single clone, and the cell population produced as a result may possibly include heterogeneous cells from plural clones of different derivation.
Therefore, the establishment of a safe human lens epithelial cell line has been desired, which is useful for the physiological and biochemical studies of the lens and the development of medicaments for the prevention and treatment of lens diseases such as cataract, and which has homogeneous properties and does not cause viral infections when grafted to living organisms.
When a lens is regenerated by grafting human lens epithelial cells into the living body, proliferation of said human lens epithelial cells need to be stopped at a certain stage and the cells should proceed to differentiation into lens fiber cells. Hence, the establishment of a human lens epithelial cell line has been simultaneously desired, which is capable of terminating the proliferation of the cells at a desired time to induce differentiation.
It is therefore an object of the present invention to provide a monoclonal human lens epithelial cell line having homogeneous property, which is useful for the physiological and biochemical studies of the lens and the development of medicaments for the prevention and/or treatment of lens diseases such as cataract, and which is free of viral infection in patients who underwent cell grafting. Another object of the present invention is to provide the above-mentioned human lens epithelial cell line capable of easy control of cell proliferation.